In the present GC/MS analysis for the derivative of sulfi def using PFBBr as a derivatization reagent, it is not necessary to extract sulfi de from blood beforehand; the method is highly
Trang 1© Springer-Verlag Berlin Heidelberg 2005
and its metabolite
By Shigetoshi Kage
Introduction
Hydrogen sulfi de ( H2S) is a colorless gas with the smell of putrid eggs; it can exist in both non-ionic and non-ionic forms in aqueous solution Th e ratio of the nonionic form to the total ionized one is infl uenced by concentration of hydrogen ion in the solution Under acidic conditions,
H2S does not ionized and evaporated from water; under alkaline conditions it is easily ionized and retained in the solution
As toxic eff ects of H2S, it (at higher than 700 ppm) acts on the central nervous system causing generalized poisoning, and also shows localized infl ammatory eff ects on the wet mucous mem-branes of the eye and respiratory organs H2S poisoning together with oxygen defi ciency is most frequent in industries; the former is also occurring at sewers, sewage treatment institutions, pe-troleum refi neries, sodium sulfi de factories, and zones of volcanos and spas Th e poisoning can also occur by ingesting a pesticide of the lime-sulfur mixture or bath salts including sulfur
It is necessary to analyze H2S in blood of a poisoned patient to verify its poisoning Th e analytical methods for H2S can be classifi ed into two groups; methods for detecting nonionic
H2S under acidic conditions and those for detecting an ionized from of H2S under alkaline conditions In this chapter, a method of GC with a fl ame photometric detector (FPD) for anal-ysis of the nonionic H2S and a method of GC/MS for the ionized form with derivatization are presented
H2S is easily oxidized to thiosulfate and sulfate in a human body [1–3] Th e levels of sulfate
in blood and urine of non-poisoned subjects are relatively high, making sulfate diffi cult to be used as an indicator of H2S poisoning However, thiosulfate can be used as the indicator of the poisoning [4–9], because its endogenous levels in human blood and urine are usually low a
Th erefore, a method for detecting this metabolite is also presented
See [10]
Preparation of the standard stock solution of H2S
i One gram of sodium sulfi de nonahydrate (Na2S · 9H2O, Wako Pure Chemical Industries, Ltd., Osaka, Japan and many other manufacturers) is placed in a volumetric fl ask (100 mL) and dissolved in purifi ed water b, which had been degassed by bubbling with nitrogen, to make 100 mL solution
ii A 25-mL volume of iodine solution [0.1 N (=0.05 M) standard solution available from Wako Pure Chemical Industries, and other manufacturers] is placed in an Erlenmeyer fl ask,
Trang 2fol-lowed by addition of 1 mL of concentrated HCl and 10.0 mL of the above Na2S · 9H2O solu-tion, and left at room temperature for 10 min
iii Th e iodine in the above solution is titrated using the titer(f)-known sodium thiosulfate solution [0.1 N=0.1 M, standard solution available from many manufacturers] in the pres-ence of the starch color reactant (1 g of starch is mixed with 10 ml water, which is put in
100 mL hot water with stirring, boiled for 1 min and cooled) using a biuret titrator
iv A volume of the sodium thiosulfate solution (0.1 M) to be required for the above titration
is assumed to be (a) mL; separately, at the step ii), 10 ml of distilled water is added in place
of 10 ml of the Na2S · 9H2O solution as a blank test and the following titration procedure is exactly the same as described above A volume of the sodium thiosulfate solution (0.1 M)
to be required for the titration of the blank test is assumed to be (b) mL
v Th e volume of Na2S · 9H2O solution prepared at the fi rst step to be used for making the fi nal standard solution of H2S is: [89.3/ (b–a)f] mL Th is volume of the solution is placed in a
100-mL volume volumetric fl ask, followed by dilution with the purifi ed water degassed with nitro-gen to make the fi nal 100 mL solution; this standard stock solution contains 152 µg/mL of H2S
GC conditions
GC: an instrument with a fl ame photometric detector ( FPD) and with a fi lter for sulfur; column:
a glass packed column (3 m × 3 mm i.d.); packing material: diatomite treated with acid and silane (60–80 mesh) and coated with 25% 1,2,3-tris(2-cyanoethoxy)propane (TCEP)c; column tem-perature: 70 °C; injection temtem-perature: 150 °C; carrier gas: nitrogen; its fl ow rate: 50 mL/min
Procedure
i One milliliter of whole blood is placed in a 10-mL volume glass centrifuge tube with a ground-in stopper
ii Five milliliters of cold acetone and 0.5 ml of 20% HCl solution are added to the above cen-trifuge tube and mixed well
iii Th e tube is centrifuged at 3,000 rpm for 5 min to remove sediment at low temperature; the supernatant fraction is decanted to another glass tube
iv Th e supernatent fraction is diluted 5–20 fold with acetone A 1–3 µL aliquot of it is injected into GC
v Using a double-logarithmic graph, a external calibration curve is drawn with H2S concen-tration (0.05–2.0 µg/mL) on the horizontal axis and with peak height (cm) on the vertical axis in advance Th e concentration (µg/mL) of H2S in a test sample is calculated using the calibration curve
Assessment of the method
When H2S in a blood specimen is extracted by the headspace method, the H2S gas in the head-space is decomposed according to heating temperature and time, resulting in variation in data obtained However, HS is relatively stable in the acetone solution acidifi ed with HCl Th e HS
Trang 3concentration in blood was measured in an H2S poisoning case by this method [11] Th e detec-tion limit is 0.1 µg/mL; the sensitivity is satisfactory However, the retention time of H2S is as short as 0.7 min; it overlaps peaks of pentane and hexane Th e retention time of acetone is 3.8 min
GC/MS analysis
See [8, 12–14]
Reagents and their preparation
• H2S standard stock solution: its preparation is the same as described in the above GC anal-ysis section
• 5 mM Tetradecyldimethylbenzylammonium chloride ( TDMBA, Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan)d / borax-saturated aqueous solution: 36.8 mg of TDMBA is dissolved in
20 mL of purifi ed water, which has been degassed with nitrogen and saturated with sodium tetraborate
• 20 mM Pentafl uorobenzyl bromide (PFBBr, GL Sciences, Tokyo, Japan and other manufac-turers) solution: 104 mg of PFBBr is dissolved in 20 mL toluene
• 10 µM 1,3,5-Tribromobenzene (TBB, Wako Pure Chemical Industries and others) solution (internal standard, IS): 31.5 mg TBB is dissolved in 100 mL ethyl acetate; the solution is diluted 100-fold with ethyl acetate
GC/MS conditions
See [8]
Column: HP-5 (30 m × 0.32 mm i.d., fi lm thickness 0.25 µm, Agilent Technologies, Palo Alto, CA, USA); column temperature: 100° C (2 min)→ 10° C/min→ 220° C (5 min); injection temperature: 220° C; ion source temperature: 210° C; carrier gas: He; its fl ow rate: 2 mL/min; injection mode: splitless; ionization mode: EI; electron energy: 70 eV; ionization current:
300 µΑ
Procedure
i A 0.8-mL volume of 5 mM TDMBA aqueous solution, 0.5 mL of 20 mM PFBBr toluene solution and 2.0 mL of 10 µM TBB ethyl acetate solution are placed in a 10-mL volume glass centrifuge tube with a ground-in stopper
ii A 0.2-mL volume of blood is added to the above mixture and vortex-mixed for 1 min
iii A 0.1-g aliquot of solid potassium dihydrogenphosphate is added to the mixture e and vortex-mixed for 10 s
iv Th e tube is centrifuged at 2,500 rpm for 5 min; the supernatant fraction is transferred to a small vial with a screw cap to serve as test solution
Hydrogen sulfi de (H2S) in blood
Trang 4v A 1-µL aliquot of the solution is injected into GC/MS.
vi A calibration curve is constructed with sulfi de concentration (µg/mL) on the horizontal
axis and with the area ratio of the peak at m/z 394 (the derivative of sulfi de) to that at
m/z 314 (IS) on the vertical axis Th e concentration of sulfi de (µg/mL) in a specimen is calculated with this curve
Assessment of the method
> Figure 2.1 shows a total ion chromatogram (TIC) and mass chromatograms for the sulfi de
derivative (retention time 9.8 min) and IS (7.0 min) [8] In the present GC/MS analysis for the derivative of sulfi def using PFBBr as a derivatization reagent, it is not necessary to extract sulfi de from blood beforehand; the method is highly sensitive, allows the fi nal identifi cation of the compound and thus is useful to verify its poisoning Since H2S is produced in putrefi ed blood and also by decomposition of cysteine [15, 16], it is necessary to construct a calibration curve
by adding sulfi de to blood obtained from healthy subjects g Th e detection limit is 0.2 µg/mL in
TIC and mass chromatograms of a derivative of sulfide obtained from blood of a victim who died
of hydrogen sulfide poisoning m/z 394: the derivative of sulfide; m/z 314: IS.
⊡ Figure 2.1
Trang 5the scan mode and 0.02 µg/mL in the SIM mode Using the present GC/MS method, the changes
in sulfi de concentration in blood during storage in a refrigerator or a freezer were reported [14, 15]; sulfi de poisoning cases were also reported [7–9, 17–19]
Toxic concentrations
In the survived cases, blood should be sampled from patients as soon as possible aft er exposure
to H2S gas, because H2S is rapidly metabolized in a human body In the experience of the author et al., sulfi de could not be detected from blood specimens sampled from six survived patients 4–15 h aft er exposure [7, 9]
> Table 2.1 summarizes H2S concentrations in blood of fatal poisoning cases Ikebuchi et al [11] detected 0.31 µg/mL of H2S from blood obtains at autopsy from a victim, who had died of poisoning by H2S gas evaporated from polluted water at an industrial waste disposal facility Kimura et al [17] autopsied 3 of 4 victims, who had died of poisoning by H2S developed from dark slime accumulated in a seawater-introducing pipe at a fl atfi sh farm, and detected 0.08– 0.5 µg/mL of sulfi de from their blood obtained Th e author et al also experienced cases, in which one subject had died by exposure to H2S gas developed from slime in an underground waste water tank of a hospital [7], in which one subject had died of H2S added for conversion
of glutathione copper into glutathione at a glutathione-refi nery factory [9], and in which one subject had died of poisoning by volcano gas fl owing backward into an oil-separating tank at a geothermal power plant [8]; the blood concentrations of sulfi de detected from these victims were 0.13–0.45 µg/mL In addition, the author et al [15] made animal experiments, in which rats were exposed to 550–650 ppm of H2S gas; the mean blood concentration of H2S in the rats (n=5) killed by H2S poisoning was 0.38 µg/mL
Th e fatal blood concentrations of sulfi de were also measured for humans and rats aft er oral ingestion of sulfi de or polysulfi deh; as shown in > Table 2.2, the concentrations of sulfi de aft er
oral ingestion were more than 20 times higher than those aft er exposure to H2S gas [18, 19]
Hydrogen sulfi de (H2S) in blood
⊡ Table 2.1
Blood concentrations of hydrogen sulfide (H 2 S) in fatal poisoning cases after exposure to its
vapor
1 Industrial waste disposal facility 0.31 [11]
2 Flatfish farm 0.08 0.50 (3 victims) [17]
3 Underground waste water tank of a hospital 0.22 [7]
4 Glutathione-refinery factory 0.13 [9]
5 Geothermal power plant 0.45 [8]
Rat experiments
(exposed to 550–650 ppm H2S)
0.38 [15]
Trang 6GC/MS analysis of thiosulfate (a metabolite of hydrogen sulfide)
in blood and urine
See [5, 8]
Reagents and their preparation
• Standard solution of sodium thiosulfate: its 0.1 M solution is commercially available (Wako Pure Chemical Industries and other manufacturers), or it can be easily prepared in a labo-ratory
• 200 mM Ascorbic acid solution: 352 mg of ascorbic acid is dissolved in purifi ed water to prepare 10 mL solution
• 5% NaCl solution: 500 mg NaCl is dissolved in purifi ed water to prepare 10 mL solution
• 20 mM Pentafl uorobenzyl bromide (PFBBr) solution: 104 mg of PFBBr is dissolved in
a cetone to prapare 20 mL solution
• 25 mM Iodine solution: 317 mg of iodine is dissolved in ethyl acetate to prepare 100 mL solution
• 40 µM 1,3,5-Tribromobenzene (TBB) solution (IS): 31.5 mg of TBB is dissolved in 100 mL ethyl acetate; 4 ml of the solution is diluted 25-fold with ethyl acetate to prepare 100 mL solution
GC/MS conditions
Column: HP-5 (30 m × 0.32 mm i.d., fi lm thickness 0.25 µm, Agilent Technologies); column temperature: 100° C (2 min)→ 10° C/min→ 220° C (5 min); injection temperature: 220° C; ion source temperature: 210° C; carrier gas: He; its fl ow rate: 2 mL/min; injection mode: splitless; ionization mode: EI; electron energy: 70 eV; ionization current: 300 µΑ
⊡ Table 2.2
Blood concentrations of sulfide in fatal poisoning cases after oral ingestion of sulfide or
polysulfide
Rat experiments
Rat experiments
Trang 7Procedure
i A 0.05-mL volume of 200 mM ascorbic acid, 0.05 mL of 5% NaCl aqueous solution and 0.5 mL of 20 mM PFBBr acetone solution are placed in a 10-mL volume glass centrifuge tube with a ground-in stopper
ii A 0.2-mL volume of blood or urinei is added to the above mixture and vortex-mixed for
1 min
iii A 2.0 mL volume of 25 mM iodine ethyl acetate solution and 0.5 mL of 40 µM TBB ethyl acetate solution are also added to the mixture and vortex-mixed for 30 s
iv Th e tube is centrifuged at 2,500 rpm for 5 min; and left at room temperature for 1 h Th en, the supernatant fraction is transferred to a small vial with a screw cap to serve as test solu-tion
v A 1-µL aliquot of the solution is injected into GC/MS
vi A calibration curve is drawn with thiosulfate concentration (µmol/mL) on the horizontal
axis and with the area ratio of the peak at m/z 426 (the derivative of thiosulfate) to that at
m/z 314 (IS) on the vertical axis Th e concentration of thiosulfate (µmol/mL) in a test spec-imen is calculated with this curve
Assessment of the method
> Figure 2.2 shows a TIC and mass chromatograms for the thiosulfate derivativej (retention time 11.9 min) and IS (7.0 min) [8] Th is method does not require any special pretreatment, and sensitive identifi cation and quantitation can be achieved like in the case of GC/MS assays
of sulfi de described before Th e detection limit was 0.02 µmol/mL in the scan mode, and 0.002 µmol/mL in the SIM mode Using the present GC/MS method, the changes in thiosulfate concentration in blood and urine during storage in a refrigerator were reported [14]; H2S poi-soning cases were also reported [7–9]
Toxic concentrations
As shown in > Table 2.3, the author et al [7] could not detect thiosulfate from blood of four
survived patients aft er exposure to H2S gas at a recycled paper manufacturing factory; the blood specimens had been sampled 6–15 h aft er the exposure However, 0.12–0.43 µmol/mL
of thiosulfate could be detected from urine in 3 of the 4 patients In a case in which 2 subjects were exposed to H2S gas during working in a close position to an instrument for exclud-ing acidic gas at an ammonia- manufacturexclud-ing factory, thiosulfate could not be detected from blood of both patients sampled 4–5 h aft er the exposure, but 0.18 and 0.50 µmol/mL thiosulfate could be detected from their urine [9] In the survived cases of animal experi-ments in which rabbits were exposed to 100–200 ppm H2S gas, 0.061 µmol/mL of thiosulfate could be detected from blood sampled just aft er the exposure, followed by a trace amount
of the metabolite 2 h aft er the exposure; while in urine of rabbits, about 1 µmol/mL of thio-sulfate could be detected 1–2 h aft er the exposure, followed by 0.51 µmol/mL 4 h aft er the exposure and further decrease according to time, but a small but higher peak of thiosulfate than the control peak could be detected even aft er 24 h [6] Th ese data show that the
measure-GC/MS analysis of thiosulfate (a metabolite of hydrogen sulfi de) in blood and urine
Trang 8⊡ Figure 2.2
TIC and mass chromatograms of a derivative of thiosulfate obtained from blood of a victim who
died of hydrogen sulfide poisoning m/z 426: the derivative of thiosulfate; m/z 314: IS.
⊡ Table 2.3
Concentrations of thiosulfate in urine of survivors after exposure to H 2 S
No Place of incident (interval between exposure
and sampling)
Concentration (µmol/mL)
Ref.
1 Recycled paper manufacturing factory
(6–15 h)
0.12–0.43 (3 victims)
[7]
2 Ammonia-manufacturing factory
(4–5 h)
0.18, 0.50 (2 victims)
[9]
(exposed to 100–200 ppm H2S for 60 min)
(exposure-to-sampling interval: 4 h)
(5 animals)
Trang 9ments of thiosulfate in urine are more eff ective than those in blood especially in survived cases
> Table 2.4 shows the thiosulfate contents in blood of fatal victims exposed to H2S gas Th e three cases are the same as those shown in > Table 2.1 [7–9] Th eir blood concentrations of thiosulfate were 0.025, 0.058 and 0.143 µmol/mL, respectively As animal experiments, rabbits were exposed to 500–1,000 ppm H2S gas until death Th e mean blood concentration of thiosul-fate in the poisoned rabbits was 0.080 µmol/mL [6] However, thiosulfate could not be detected from rabbit urine, probably because of their sudden death due to exposure to H2S It can be thus concluded that the measurements of thiosulfate in blood are more eff ective than those in urine for such sudden death cases
Notes
a) Kawanishi et al [20] analyzed thiosulfate in urine and plasma of 5 healthy subjects; thio-sulfate concentrations in urine and plasma were 31.2 µmol/24 h (0.0288 µmol/mL) and 0.00268 µmol/mL, respectively Th e author et al [5] also detected 0.007 µmol/mL (mean value) of thiosulfate from urine of 12 healthy subjects; while the level in blood was below the detection limit (0.003 µmol/mL)
b) Since H2S can be decomposed by oxygen dissolved in water, the purifi ed water degassed with nitrogen gas is used Th e purifi ed water aft er boiling, followed by cooling to room temperature, can be also used
c) A similar packing material can be purchased from GL Sciences, Tokyo, Japan
d) Th e reagent is a quaternary ammonium compound to be used as a phase-transfer-catalyst Another group reported a polymer-bound tributylmethylphosphonium chloride for such a type of catalysis [13]
e) Under alkaline conditions, sulfur-containing compounds, such as cysteine and glutathione,
in blood decompose to produce sulfi de To suppress these reactions, the pH of the mixture
is made acidic
f) Th e derivatization reaction of sulfi de is:
2R-Br + Na2S → R-S-R + 2NaBr
R = pentafl uorobenzyl
g) McAnalley et al [21] analyzed blood sulfi de for 100 subjects without any exposure to H2S; the results were not greater than 0.05 µg/mL Th e author et al [15] found that the blood sulfi de levels were markedly infl uenced by postmortem intervals and by temperatures of specimens
GC/MS analysis of thiosulfate (a metabolite of hydrogen sulfi de) in blood and urine
⊡ Table 2.4
Concentrations of thiosulfate in blood after death by H 2 S poisoning
(µmol/mL)
Ref.
1 Underground waste water tank of a hospital 0.025 [7]
2 Glutathione-refinery factory 0.058 [9]
3 Geothermal power plant 0.143 [8]
Rabbit experiments (exposed to 500–1,000 ppm H2S) 0.080 [6]
Trang 10for storage When blood specimens are sampled within 24 h aft er death and stored at not higher than 20° C, the postmortem production of H2S can be suppressed; the sulfi de concen-tration in blank blood was not greater than 0.01 µg/mL When the specimens are stored in a refrigerator or in a freezer, the postmortem production of H2S due to putrefaction could be suppressed even for the blood specimens sampled from a cadaver with a postmortem interval
of more than 24 h
h) When polysulfi de is ingested orally, the unchanged compound can be detected from blood [18]
i) Blood is the suitable specimen for fatal poisoning cases; while urine is suitable for survived cases aft er poisoning
j) Th e derivatization reaction for thiosulfate is shown as follows It consists of two-step reac-tions; the fi rst one is alkylating reaction and the second one oxidation reaction
Alkylating reaction:
R-Br + Na-S-SO3Na → R-S-SO3Na + NaBr
R = pentafl uorobenzyl
Oxidation reaction:
2R-S-SO3Na + I2 + 2H2O
→ R-S-S-R + 2NaHSO4 + 2HI
References
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2) Bartholomew TC, Powell GM, Dodgson KS et al (1980) Oxidation of sodium sulphide by rat liver, lungs and kidney Biochem Pharmacol 29:2431–2437
3) Beauchamp RO, Bus JS, Popp JA et al (1984) A critical review of the literature on hydrogen sulfide toxicity Crit Rev Toxicol 13:25–97
4) Kangas J, Savolainen H (1987) Urinary thiosulfate as an indicator of exposure to hydrogen sulphide vapour Clin Chem Acta 164:7–10
5) Kage S, Nagata T, Kudo K (1991) Determination of thiosulfate in body fluids by GC and GC/MS J Anal Toxicol 15:148–150
6) Kage S, Nagata T, Kimura K et al (1992) Usefulness of thiosulfate as an indicator of hydrogen sulfide poisoning
in forensic toxicological examination: a study with animal experiments Jpn J Forensic Toxicol 10:223–227 7) Kage S, Takekawa K, Kurosaki K et al (1997) The usefulness of thiosulfate as an indicator of hydrogen sulfide poisoning: three cases Int J Legal Med 110:220–222
8) Kage S, Ito S, Kishida T et al (1998) A fatal case of hydrogen sulfide poisoning in a geothermal power plant J Forensic Sci 43:908–910
9) Kage S, Kudo K, Ikeda N (1998) Determination of sulfide, thiosulfate and polysulfides in biological materials for diagnosis of sulfide poisoning Jpn J Forensic Toxicol 16:179–189 (in Japanese with an English abstract) 10) Tanaka E, Nakamura T, Terada M et al (1987) Determination of hydrogen sulfide in fluid and organ specimens
by gas chromatography with a flame photometric detector Eisei Kagaku 33:149–152 (in Japanese with an English abstract)
11) Ikebuchi J, YamamotoY, Nishi K et al (1993) Toxicological findings in a death involving hydrogen sulfide Jpn J Legal Med 47:406–409 (in Japanese with an English abstract)
12) Kage S, Nagata T, Kimura K et al (1988) Extractive alkylation and gas chromatographic analysis of sulfide J Forensic Sci 33:217–222
13) Miki A, Tsuchihashi H (1999) Determination of hydrogen sulfide in blood by gas chromatography/mass spec-trometry after liquid-liquid-solid phase-transfer-catalyzed pentafluorobenzylation Jpn J Forensic Toxicol 17: 14–